Bacterial pathogens must rapidly adapt to limiting nutrients during infection. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved phosphorelay that couples sugar transport with phosphorylation and serves as a monitor of carbohydrate flow in the bacterial cell. The Group A Streptococcus (GAS) is a significant human-restricted pathogen causing a wide array of acute diseases in different host tissues, resulting in over half a million deaths worldwide each year. Relevant to this renewal, GAS and other pathogenic streptococci depend upon carbohydrate uptake systems for their ability to survive in the host. Mga regulates virulence genes encoding immune evasion factors, adhesins, and sugar utilization operons, and we have established over the previous grant period that Mga is directly phosphorylated by the PTS at conserved histidines within PTS regulatory domains (PRD) that alter Mga-regulated gene expression and virulence in GAS. Paralogs exist in GAS (RofA-like proteins, RALPs) as well as orthologs in other G+ pathogens such as S. pneumoniae (MgaSpn) and B. anthracis (AtxA). Mga, with AtxA, form the paradigms for a family of PRD- Containing Virulence Regulators (PCVRs) in G+ pathogens that that allow sugar availability to be sensed by global virulence regulatory pathways and influence the disease process. Based on our substantial published data, we posit that as GAS encounter changing or limiting sugar sources in the host, the PTS phosphorylates Mga in PRD-1 to block dimerization and inhibit its activity, effectively shutting Mga off. Our molecular dissection of the PTS in GAS has shown that glucose levels significantly alter PTS-mediated Mga phosphorylation and the Mga regulon. However, many questions still remain. The goal of this renewal will be to continue our pioneering studies on Mga as an archetype PCVR, while expanding our scope to other putative PCVRs. In Aim 1, we will delineate how glucose uptake in GAS impacts Mga by defining the PTS and non- PTS components for glucose uptake that influence Mga phosphorylation and activity using state-of-the-art LC-MS/MS approaches to monitor phosphohistidines. In Aim 2, we will determine how non-glucose PTS sugars signal through Mga in GAS using growth in PTS sugars with specific PTS EIIC mutants complemented with Tn-seq to map Mga-specific genetic interactions in different PTS sugars. Aim 3 will focus on whether paralogs and homologs of Mga are functional PCVRs by exploring whether the PTS directly phosphorylates and influences the activity of paralogs in GAS (RofA, RivR), homologs in S. dysgalactiae (DmgB) and S. pneumoniae (MgaSpn). Finally, Aim 4 will investigate the role of PTS regulation on Mga and RALP paralogs in two host environments representing glucose levels known to affect Mga; bloodstream infection (high glucose) using ex vivo and in vivo models, and mucosal colonization (low glucose) using a murine IL17-/- NALT model. Advancing our understanding of PCVRs could lead to novel strategies to treat severe infections caused by these important pathogens.